Terrestrial vertebrates protect themselves from chemical and physical damage and uncontrolled water loss by maintaining a barrier in their epidermis. In mammals this is accomplished by forming a highly insoluble protein and lipid structure on the surface of the corneocytes termed the cornified envelope (CE) and by impeding water diffusion across the stratum corneum by mortaring together the corneocytes by layers of hydrophobic skin specific lipids. (Downing et al., Dermatology in General Medicine, Fitzpatrick, et al., eds., pp. 210-221 (1993) and Ponec, M., The Keratinocyte Handbook, Leigh, et al., eds., pp. 351-363 (1994)). These lipids differ in composition from other bilayer forming lipids found in all living cells. Notably they have a diminished phospholipid content and, instead, contain increased amounts of cholesterol, its acyl and sulfate esters, fatty acids, and several epidermal specific long chain hydroxy- and hydroxyacyl (glucosyl)-ceramides. (Elias, P. M. and G. K. Menon, Skin Lipids, Advances in Lipid Research, Vol. 24, pp. 1-26 (1991)).
Synthesis of the lipids that join the CE is initiated in the spinous layer. These lipids are temporarily stored in lamellar bodies of stratum granulosum, where they are arranged as stacks of tetralaminar sheets. Preceding or paralleling the formation of the protein barrier of the CE, the contents of the lamellar bodies are extruded into the intercellular space. One component of these lipids are epidermal specific long chain ω-hydroxyceramides, which become covalently attached onto the outer surface of the CE as a ˜5 nm monomolecular layer. One idea is that these protein-linked ceramides interdigitate with the intercellular lipid in a comb-like fashion, presumably to stack them into ordered lamellae. (Wertz, P. W., Experientia Suppl., 78: 227-237 (1997) and Wertz, P. W. and D. T. Downing, Physiology, Biochemistry and Molecular Biology of the Skin, Goldsmith, L. A., ed., Vol. 1, pp. 205-236 (1991)).
Many icthyosiform diseases (e.g., autosomal recessive lamellar ichthyosis and recessive congenital nonbullous ichthyosiform erythroderma) are caused by improper or incomplete lipid barrier function. For example, Lamellar Ichthyosis (LI) is a clinically heterogeneous autosomal recessive disorder, which causes abnormalities of the CE and persons with the severe LI phenotype often present at birth encased in a translucent colloidion membrane. Soon after birth, this thick membrane dries and cracks and, over time, these persons develop large, brown, plate-like scales in a generalized distribution. (Russell et al., Nat. Genet. 9:279 (1995)).
The locus for LI has been mapped to chromosome 14q11 and a complete linkage with the gene encoding transglutaminase 1 (TGase 1) was found. (Russell et al., Nat. Genet. 9:279 (1995)). Further, point mutations in TGase 1 were identified in two of the multiplex LI families used in the linkage study. (Russell et al., Nat. Genet. 9:279 (1995)). These mutations are hypothesized to adversely affect the formation of cross-links essential in the production of the CE. (Russell et al., Nat. Genet. 9:279 (1995)).
To form a healthy CE, specialized keratinocyte proteins are expressed and subsequently made insoluble by cross-linking by both disulfide bonds and isopeptide bonds formed by transglutaminases (TGases). (Matoltsy, A. G. and M. N. Matoltsy, J. Invest Dermatol., 46: 127-129 (1966); Sun, T-T. and H. Green, Cell, 9: 511-521 (1976); Greenberg, et al., FASEB J., 5: 3071-3077 (1991); Reichert, et al., Molecular Biology of the Skin, Darmon, M. and M. Blumenberg eds., pp. 107-150 (1993); and Simon, M., The Keratinocyte Handbook, Leigh, et al., eds., pp. 275-292 (1994)). Several investigators believe that the protein composition of CEs varies widely between epithelia and even different body sites of epithelia such as the epidermis. (Steinert, et al. J. Biol. Chem., 273: 11758-11769 (1998) and Song, et al., Genomics, 55:28-42 (1999)). However, involucrin seems to be a ubiquitous component of most if not all CEs. (Simon, M., The Keratinocyte Handbook, Leigh, et al., eds., pp. 275-292 (1994) and Steinert, P. M. and L. N. Marekov, J. Biol. Chem., 272: 2021-2030 (1997)).
The function of involucrin can be characterized as that of a protein scaffold since it is one of the first proteins deposited at or near the membrane surface in the vicinity of desmosomes to initiate CE formation and is subsequently joined to several reinforcement proteins, as well as, ceramide lipids. (Steinert, P. M. and L. N. Marekov, J. Biol. Chem., 272: 2021-2030 (1997); Rice, R. H. and H. Green, Cell, 18: 681-694 (1979); Simon, M. and H. Green, Cell, 36: 827-834 (1984); Yaffe et al., J. Invest. Dermatol., 100: 3-9 (1993); Crish et al., Differentiation, 53: 191-200 (1993); Steinert, P. M., Cell Death Different., 2: 33-40 (1995); Ishida-Yamamoto, et al., J. Invest. Dermatol., 108: 12-16 (1997); Candi et al., Proc. Natl. Acad. Sci. USA, 95: 2067-2072 (1998)); and (Marekov, L. N. and P. M. Steinert, J. Biol. Chem., 273: 17763-17770 (1998)). Expression studies, for example, demonstrate that involucrin deposition at the cell periphery precedes all other suspected or confirmed CE protein constituents. (Rice, R. H. and H. Green, Cell, 11: 417-422 (1979); Watt, F. M. and H. Green, J. Cell Biol., 90: 738-742 (1981); Simon, M. and H. Green, Cell, 36: 827-834 (1984); Simon, H. and H. Green, J. Biol. Chem., 263: 18093-18098 (1988); Yaffe, et al., J. Biol. Chem., 267: 12233-12238 (1992); Yaffe, et al., J. Invest. Dermatol., 100: 3-9 (1993); Crish, et al., Differentiation, 53: 191-200 (1994); and de Viragh, et al., J. Invest. Dermatol., 103: 815-819 (1994)).
Additionally, shadowing and scanning transmission electron microscopy have revealed that a monomolecular layer of involucrin is overlayered on the cytoplasmic side by other CE structural proteins. (Jarnik, et al., J. Cell Sci., 111: 1051-1060 (1998)). Further, extant models of CE structure based on biochemical characterization and protein sequencing indicate that involucrin becomes cross-linked to several reinforcement CE proteins including other molecules of involucrin, trichohyalin, elafin, repetin, periplakin, desmoplakin, envoplakin, keratin intermediate filaments, members of the small proline rich family, cystatin α, and loricrin. (Steinert, et al. J. Biol. Chem., 273: 11758-11769 (1998); Steinert, P. M. and L. N. Marekov, J. Biol. Chem., 272: 2021-2030 (1997); and Robinson, et al., J. Biol. Chem., 272: 12035-12036 (1997)). Still further, recent data have shown that involucrin is a major target for the covalent attachment of ceramide lipids from the exterior surface of the CE, which could only occur if involucrin was deposited in the intimate vicinity of the keratinocyte membrane at an early time. (Marekov, L. N. and P. M. Steinert, J. Biol. Chem., 273: 17763-17770 (1998)).
Human involucrin contains 150 glutamine and 45 lysine residues and it appears that mammalian involucrins have undergone extensive expansion of various glutamine-rich repeating motifs during evolution perhaps to increase the sites suitable or available for TGase mediated cross-linking. (Eckert, R. L. and H. Green, Cell, 46: 583-589 (1988) and (Tseng, H. and H. Green, Cell, 54: 491-496 (1988)). Indeed, involucrin is an excellent substrate for transglutaminases (TGases) both in vitro and in vivo. (Simon, M. and H. Green, J. Biol. Chem., 263: 18093-18098 (1998); Etoh et al., Biochem. Biophys. Res. Commun., 136: 51-56 (1986); and (Steinert, P. M. and L. N. Marekov, J. Biol. Chem., 272: 2021-2030 (1997)). Sequencing studies have shown, however, that only a limited number of these residues are used for cross-linking in vivo. (Steinert, P. M. and L. N. Marekov, J. Biol. Chem., 272: 2021-2030 (1997)).
The human TGase 1 gene (TGase 1) encodes a 92-kDa protein consisting of 816 amino acid residues located on chromosome 14q11.2. (Kim et al., J. Bio. Chem. 267:7710-7717 (1992)). TGases are Ca2+-dependent enzymes, which catalyze the transfer of the γ-carboxyl group from protein-bound glutamine to the ε-amino group of protein bound lysine residues or other primary amines. These enzymes are responsible for the cross-linking of CE proteins into a chemically and mechanically resistant protein polymer. (Greenberg et al., FASEB J., 5: 3071-3077 (1991); Reichert et al., Molecular Biology of the Skin The Keratinocyte, Darmon et al., eds., pp. 107-150 (1993); and Melino et al., Meth. Enzymol., in press (1999)). Of the seven known human TGases, four (TGases 1, 2, 3 and X) are expressed in terminally differentiating epithelia such as the epidermis. (Aeschlimann et al., J. Biol. Chem., 273: 3452-3460 (1998) and Kim et al., J. Biol. Chem., 266: 536-539 (1991)). Of these, the TGase 2 enzyme is thought to play only a minor role, and the properties of the newly discovered TGase X enzyme await characterization.
The TGase 1 and 3 enzymes are essential for the cooperative cross-linking of such substrates as loricrin, trichohyalin, and small proline rich proteins 1 and 2. (Candi, et al., J. Biol. Chem., 274:7226-7237 (1999); (Tarcsa, et al., J. Biol. Chem., 272: 27893-27901 (1997); (Candi, et al., J. Biol. Chem., 270: 26382-26390 (1995); and (Tarcsa, et al., J. Biol. Chem., 273: 23297-23303 (1998)). The TGase 3 enzyme is soluble and requires proteolytic activation before it can function. (Kim, et al., J. Biol. Chem., 265: 21971-21978 (1990)). The TGase 1 enzyme was first discovered in keratinocytes and is usually anchored to membranes by way of acyl N-myristoyl and S-myristoyl or S-palmitoyl adducts near the amino terminus of the protein. (Chakravarty, R. and R. H. Rice, J. Biol. Chem., 264: 625-629 (1989); Phillips, et al., Biochemistry, 32: 11057-11063 (1993); and Steinert, et al., J. Biol. Chem., 271: 26242-26250 (1996)). While involucrin appears to be a good substrate for TGases in in vitro reactions, extant data have provided no information on which of these enzyme(s) are responsible for cross-linking in vivo.
Clearly, several crucial pieces of the puzzle are missing and a better understanding of CE assembly and ceramide lipid attachment would enable the development of novel biotechnological tools, therapeutics, prophylactics and cosmetics for the study, treatment and prevention of icthyosiform diseases and other related skin disorders.